The cellular expression of immune response (Ir) gene function was studied in both primary and secondary in vitro antibody responses to the TNP conjugates of (T,G)-A--L and (H,G)-A--L. The function of accessory cells in responses to TNP-(T,G)-A--L and TNP-(H,G)-A--L is under the control of genes which also map to I-A. In contrast, the expression of Ir gene function by B cells is related to the B cell activation pathway; Ir gene function is expressed by B cells activated under conditions involving MHC-restricted T-B interaction. In vitro augmented primary and secondary responses to TNP-nuclease (TNP-NASE) have also been established and documented to be under the control of H-2 linked Ir gene(s) mapping to the I-B subregion. For these responses, accessory cell function was shown to be under Ir gene control. Recent data employing monoclonal anti-Ia reagents have suggested that genes in the I-A subregion may also be involved in regulating responses to TNP-NASE. In order to further analyze the genetic regulation of T cell responses to NASE, a series of cloned lines were generated in BALB/c (H-2d) as well as (H-2b x H-2a)F1 T cells. Individual BALB/c clones were restricted to recognizing NASE in the context of either I-Ad or I-E products. Individual F1 clones were specific for NASE in association with either H-2a or H-2b antigen-presenting cells and subregion mapping studies are currently in progress. The antigen fine specificity of cloned NASE-specific T cells is also being probed through the use of mutant NASE molecules, synthetic peptides corresponding to segments of NASE, and monoclonal antibodies specific for different determinants on the NASE molecule.